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Biologisten makromolekyylien kolmiulotteisten rakenteiden määrittäminen liuostilassa. Nobel. 2002. Kurt Wüthrich. Proteiinien NMR-spektroskopia. Primaarirakenteen määrittäminen Sekundaari ja tertiäärirakenteen tai konformaation määrittäminen Kinetiikan ja molekulaarisen liikkeen tutkimus

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biologisten makromolekyylien kolmiulotteisten rakenteiden m ritt minen liuostilassa
Biologisten makromolekyylien kolmiulotteisten rakenteiden määrittäminen liuostilassa

Nobel

2002

Kurt Wüthrich

proteiinien nmr spektroskopia
Proteiinien NMR-spektroskopia
  • Primaarirakenteen määrittäminen
  • Sekundaari ja tertiäärirakenteen tai konformaation määrittäminen
  • Kinetiikan ja molekulaarisen liikkeen tutkimus
  • Molekulaaristen vuorovaikutusten tutkimus
proteiinin nmr spektri 1957
Proteiinin NMR-spektri1957

Nobel

E. Purcell

1952

Felix Bloch

bovine pancreatic ribonuclease

Saunders, Wishnia and Kirkwood

nmr spektroskopian perusajatus
NMR-spektroskopian perusajatus

Jokainen atomi, jolla on magneettinen ydin,

antaa yksittäisen signaalin,

joka sisältää informaatiota

paikallisesta kemiallisesta ympäristöstä,

rakenteesta ja dynamiikasta.

magneettinen ydin
Magneettinen ydin

Jokainen atomi, jolla on magneettinen ydin ....

Stabiileja isotooppeja

1H, ~100%

13C, ~1.1%

15N, ~0.4%

31P, ~100%

2H, ~ 0%

Pieni magneettinen momentti

~ sauvamagneetti

miten valmistan nmr n ytteen
Miten valmistan NMR-näytteen?

1H, 31P-leimaus sellaisenaan

  • 13C, 15N-leimaus
  • Proteiinit: tuotetaan rikastetuista lähtöaineista:
  • 13C-glukoosi, 15N-ammonium suolat
  • DNA: PCR (lyhyet ketjut)
  • RNA: In vitro synteesi
  • 2H-leimausproteiini tuotetaan 2H-rikastetussa
  • mediumissa
proteiinin nmr spektri
Proteiinin NMR-spektri

... yksittäinen signaali ...

paikallinen kemiallinen magneettinen ymp rist
Paikallinen kemiallinen (~magneettinen) ympäristö
  • NMR-spektrometrin kenttä
  • paikallinen kenttä
  • -lähellä olevat ytimet
  • -ympäröivät elektronipilvet
spektroskopian perusteista
Spektroskopian perusteista

Energia kaavio – Purcellin kuva

B/E

resonanssispektroskopia
Resonanssispektroskopia

B

aika

Fourier

muunnos

Vektorimalli – Blochin kuva

taajuus

kaksiulotteinen korrelaatiospektroskopia

f1

t1

t2

f2

Kaksiulotteinen korrelaatiospektroskopia

Signaalin modulaatio ajan t1 kuluessa

S(t1, t2)

S(f1, f2)

 FT 

Aikaulottuvuus  Fourier muunnos  taajuusulottuvuus (spektri)

nmr spektroskopia kahdessa ulottuvuudessa
NMR-spektroskopia kahdessa ulottuvuudessa

1991

Richard Ernst

Jean Jeener

nmr of biological macromolecules multidimensional multinuclear spectroscopy

NMR of Biological MacromoleculesMultidimensional Multinuclear Spectroscopy

Structural Biology

how to interpret spectra
How to Interpret Spectra?

?

  • Structural implications
  • Atom type (and near neighbours)
  • Spatially near neighbours
  • Chemically bonded neighbours
  • Dynamic consequences
  • Fluctuating magnetic environment
  • Spectral parameters
  • Resonance frequency
  • Modulation of frequency
  • Correlation via dipolar field
  • Correlation indirectly via electrons (scalar coupling)
  • Relaxation
magnetic environment dispersion of resonances
Magnetic EnvironmentDispersion of resonances
  • External magnetic field of the NMR-spectrometer
  • Local fields due to
  • -adjacent nuclei
  • -surrounding electron clouds
  • Chemical shift
  • = g(1 - s)B

s is shielding (tensor)

assignment of resonances
Assignment of Resonances

Proteins display large dispersion

because they contain distinct

magnetic microenvironments.

assignment of resonances identification of residues by characteristic chemical shifts
Assignment of ResonancesIdentification of Residues by Characteristic Chemical Shifts

Aliphatic carbon shifts are particularly

characteristic for the residues.

assignment of backbone resonances principle sequential walk
Assignment of Backbone ResonancesPrinciple – Sequential Walk

HNCA

H R H R H R

| | | | | |

-N–Ca– C –N– Ca– C –N– Ca– C-

| || | || | ||

H O H O H O

Ca

S( Hi, Ni, Cai, Cai-1 )

N

H

HN(CO)CA

H R H R H R

| | | | | |

-N–Ca– C –N– Ca– C –N– Ca– C-

| || | || | ||

H O H O H O

Ca

S( Hi, Ni, Cai-1 )

N

H

slide20

HNCA

HN(CO)CA

slide22

The redundancy in many alternatives for sequential assignment

is important for automated assignment.

spectra contain implict structural data noes short distances
Spectra Contain Implict Structural DataNOEs  Short Distances

NOEs

Nuclear Overhauser

Enhancement

i.e. dipole-dipole

relaxation.

slide24

Short Range Distances (NOEs)

ri = rref(Sref/Si)1/6

how to convert spectral parameters to explicit structural data
How to Convert Spectral Parameters to Explicit Structural Data?
  • Short (<5-7Å) distances
  • via nuclear Overhauser spectroscopy (NOE)
  • Torsion angles
  • via scalar couplings (J-couplings)
  • Angles
  • via residual dipolar couplings (RDC)
  • Hydrogen bonds
  • via correlation spectroscopy
  • Secondary structures
  • via chemical shifts (resonance frequences)
computation of structure

T

t

Computation of Structure

Conversion of structural data to restraints

expressed as pseudo potentials

Restrained molecular dynamics (MD)

(Cartesian or torsion angle)

result family of structures
Result – Family of Structures

All structures that satisfy restraints

(within experimental error) are possible.

evaluation of structure
Evaluation of Structure
  • Accuracy
  • Restraint violations
  • Inconsitancies
  • Ramachandran violations
  • Precision
  • Spread of the family
  • Number of restraints
  • per residue
direct inspection of spectra
Direct Inspection of Spectra

Observing binding

Mapping binding epitopes

Detecting

conformational changes

about field fluctuations
About Field Fluctuations

”Reasons”

”Spectral Manifestations”

  • Bond vibrations
  • from pico to nano seconds
  • Conformational changes
  • from micro to milli seconds
  • Chemical exchange
  • from micro seconds to days
  • Relaxation measurements
  • -> rate constants, order
  • parameters, correlation times
  • Relaxation measurements
  • -> dispersion of parameters
  • Line width analysis
  • -> rate constants

Motional model

relaxation dispersion
Relaxation Dispersion

Transverse relaxation rates

vs. effective field and temperature

Frans A.A. Mulder et al.Nature Structural Biology 8, 932 - 935 (2001)

hydrogen exchange
Hydrogen Exchange

Monitoring signal intensity

after dissolving to D2O

Denis Canet et al.Nature Structural Biology - Published online: 11 March 2002,

hydrogen exchange1
Hydrogen Exchange

Denis Canet et al.Nature Structural Biology - Published online: 11 March 2002,

reaction dynamics
Reaction Dynamics

Elan Zohar Eisenmesser,1 Daryl A. Bosco,1 Mikael Akke,2 Dorothee Kern1*

Science - Feb 2002,

net alignment

Vfree

Net Alignment

Vrestricted

D = Dmax(3cos2q-1)/2